Covalent modification of histones play important roles in regulating chromatin dynamics and transcription (Jenuwein et al., (2001) Science 293:1074-80; Zhang et al., (2001) Genes Dev 15:2343-60). One example of histone modification is ubiquitination, which mainly occurs on histones H2A and H2B (Zhang, (2003) Genes Dev 17:2733-40). Although recent studies have uncovered the enzymes involved in histone H2B ubiquitination (Robzyk et al., (2000) Science 287:501-4; Hwang et al., (2003) Mol Cell 11: 261-6; Wood et al., (2003) Mol Cell 11:267-74) and a “cross-talk” between H2B ubiquitination and histone methylation (Sun et al., (2002) Nature 418:104-8; Dover et al., (2002) J. Biol. Chem. 277:28368-71), the responsible enzymes and the functions of H2A ubiquitination are unknown.
Ligation of ubiquitin involves a series of enzymatic reactions. First, ubiquitin itself is activated with ATP by the ubiquitin-activating enzyme (E1), then transferred to a ubiquitin carrier protein (E2), and finally it is transferred to ubiquitin ligase (E3). E3 enzyme catalyzes the covalent modification of lysine residues of the target protein with activated ubiquitin (Chau et al. (1989) Science 243:1576-1583). Recently, an additional conjugation factor, named E4, has been shown to be important in the ubiquitination process in conjunction with E1, E2, and E3 (Koegl et al. (1999) Cell 96(5):635-44).
The E1 and E2 ubiquitin ligases are structurally related and well characterized enzymes. There are several species of E2, some of which act in preferred pairs with specific E3 enzymes. E3 enzymes contain two separate activities: a ubiquitin ligase activity to conjugate ubiquitin to substrates, and a targeting activity to physically bring the ligase and substrate together. Substrate specificity of different E3 enzymes is the major determinant that confers specificity for different target proteins.
The inventors now report the purification and functional characterization of a ubiquitin E3 ligase complex.